The present invention relates generally to nanofluidic techniques. In particular, the invention provides a method and system for computing copy number variation in a DNA sample using digital PCR. More particularly, the present method and system partitions a DNA sample into a number of separate reaction chambers present in a nanofluidic chip forming a digital array. Merely by way of example, the nanofluidic methods and systems described herein are used to determine accurate estimates for concentrations of target gene and reference gene molecules in a biological sample as well as ratios of the determined concentrations. Although the techniques for nanofluidic systems are applied to digital PCR using digital arrays, it would be recognized that the invention has a much broader range of applicability.
Some conventional digital polymerase chain reaction (PCR) techniques utilize sequential limiting dilutions of target DNA followed by amplification using PCR. Using this digital PCR technique, it is possible to quantitate single DNA target molecules. Another digital PCR technique utilizes a microfluidic biochip in which DNA molecules are partitioned rather than diluted. As an example, the microfluidic biochip typically utilizes integrated channels and valves that partition mixtures of sample and reagents into reaction chambers having nanoliter volumes. DNA molecules in each mixture are randomly partitioned into the various chambers of the biochip, the chip is thermocycled, and the chip is imaged to determine the number of reaction chambers having a desired DNA molecule.
Copy number variation (CNV) is the gain or loss of genomic regions which range from 500 bases on upwards in size. Whole genome studies have revealed the presence of large numbers of CNV regions in human DNA and a broad range of genetic diversity among the general population. Copy number variations have been the focus of a number of recent studies as a result of their role in human genetic disorders.
Current whole-genome scanning technologies use array-based platforms (array-CGH and high-density SNP microarrays) to study CNVs. These approaches are characterized by high throughput, but lack resolution and sensitivity. Real-time PCR is a sequence-specific technique that is easy to perform, but is limited in its discriminating power beyond a 2-fold difference. CNV determination using a digital array is based upon the ability to partition DNA sequences. Given the number of molecules per panel and the dilution factor, the concentration of the target sequence in a DNA sample can be accurately calculated.
In a multiplex PCR reaction with 2 or more assays, multiple genes can be quantitated simultaneously and independently, effectively eliminating any pipetting errors if separate reactions have to be set up for different genes. When a single copy reference gene (e.g., RNase P) is used in the reaction, the ratio of the target gene to the reference gene would reflect the copy number per haploid genome of the target gene.
DNA quantitation in the digital array is based on the partitioning of a PCR reaction into an array of several hundreds or even a few thousands of reaction chambers or wells. If a DNA sample only includes several DNA molecules of interest in the sample, most of the reaction chambers in a digital PCR chip will include either one or no molecules. Thus, to first order, the number of positive reaction chambers at the PCR end-point provides a count of the molecules of interest in the sample. However, if the number of molecules of interest is large compared to the number of reaction chambers, it is likely that a number of the reaction chambers will include more than one molecule of interest and the positive reaction chamber count will be significantly less than the number of molecules of interest. Thus, there is a need in the art for improved methods and systems for estimating the number of molecules in a DNA sample.